Solution gradients or density gradients are utilized in bio-chemical research to separate macromolecules such as proteins, DNA and RNA, and larger aggregates such as viruses and cells.
Solution gradients usually utilize a solute of varying concentrations to aid in the separation of particles. Examples of appropriate solutes are: sucrose, CsCl, Percoll.TM., ficoll, metrizamide, Nycodenz.TM., sodium acetate and/or glycerol. Particles are separated either by their velocity of sedimentation in a centrifugal field, or by their density in a centrifugal field if there is an isopycnic point within the solution column in the tube. Faster, or denser particles will appear lower in the tube.
After the sample has been subjected to the centrifuge, the particles are recovered from the gradient for analysis. Fractionation methods and apparatus used to recover the sample in the gradient involve the transfer of the entire gradient or certain layers or bands of the solution gradient to other vessels. It is often desired to extract only desired bands from the solution gradient for electron microscopy, liquid scintillation or gel electrophoresis.
One of the earliest and simplest methods of fractionation is to pierce the bottom of the centrifuge tube with a fine beveled needle and collect the drops of the solution gradient as it flows through the needle into a second vessel. The flow of the solution into the opening of the needle becomes conical. In other words, the particles directly in front of the needle opening and within the cone are drawn into the needle opening before particles outside the cone. The resulting fractionation of layers of the solution gradient does not have a high resolution.
Side hole needles have also been used for fractionation. Side hole needles have a hole on each side of the needle tip. Side hole needles are more effective than the beveled needle, but side hole needles also draw the solution into the needle in a conical fashion preventing high resolution of the fractionation.
Another method for fractionating solution gradients introduces a dense solution at the bottom of a holding tube which floats the gradient up to an inverted collection funnel. The vertical movement of the gradient causes contamination of some fractions with particles retarded near the wall of the holding tube, again preventing high resolution of the fractionation.
In U.S. Pat. No. 4,003,834 to Coombs, issued Jan. 18, 1977, an apparatus is disclosed for the fractionation of a solution gradient by displacement with a piston. The piston has a collection tip, which has a biconcave conical face, at its distal end. The tip has twelve collection holes about the surface of the tip. The piston is inserted into the centrifuge tube, urged downward against the solution, displacing the solution upwardly through the piston through the collection holes in the tip into a collection tube for transferring to another vessel. A rinse tube is disposed within the piston to allow for cleaning of the tip. Air pressure may be pumped into the piston through the rinse tube to transfer any solution gradient left in the piston to the second vessel.
While the apparatus disclosed in U.S. Pat. No. 4,003,834 has improved the resolution of the fractionation of a solution gradient, some mixing of the layers is still apparent during fractionation. Furthermore, the rinsing system of the U.S. Pat. No. 4,003,834 is susceptible to contamination during fractionation. Both factors contribute to limit the resolution of the fractionation.